Hepatitis B virus (HRV) causes both acute and chronic diseases and poses a worldwide public health problem. HBV manifests itself as a chronically debilitating infection which can result in progressively severe liver damage, primary carcinoma and death. In the majority of cases, patients completely recover from HBV. However, a significant segment of the population which is infected with HBV becomes chronic carriers of the disease with the potential of transmitting the disease to others.
Recent advances in recombinant DNA techniques have provided many useful methods for elucidating the genetic structure of the HBV, as well as providing the means for preparing vaccines against HBV. The HBV genome is now known to consist of approximately 3.2 kilobase pairs of partially double stranded DNA with a DNA polymerase covalently attached enclosed in a 27 nm nucleocapsid. The nucleocapsid is enveloped in a lipoprotein coat consisting of cellular lipids and hepatitis B surface antigens (HBsAg); this is called the virion and is 42 nm in diameter.
It has also been discovered that the vital coat consists of three different but related surface proteins. These proteins are referred to generally as S, PreS.sub.2 and PreS.sub.1 proteins. Each virion is comprised of 300-400 S protein molecules and 40-80 preS.sub.2 and preS.sub.1 protein molecules.
The S protein consists of 226 amino acids and is the major component of normal vital lipoprotein coat. The S protein is approximately 24-25 kilodalton (kDa), and may be referred to as P24 or P25. The S protein may also be glycosylated to a 27-28 kilodalton glyco-protein referred to as GP27 or GP28.
The second HBsAg protein is the PreS.sub.2 surface antigen, also referred to as the middle HBsAg polypeptide. PreS.sub.2 consists of 281 amino acids formed by the addition of 55 amino acids to the N-terminus of the S protein. The PreS.sub.2 protein is approximately 31 kilodaltons and may be referred to as the P31 protein. The PreS.sub.2 protein also has two glycosylated forms, 33 kilodaltons and 36 kilodaltons, referred to respectively as GP33 and GP36. This antigen is thought to elicit an additional antigenic response in persons who do not respond to S or who respond weakly to S.
The third HBsAg protein is the PreS.sub.1 surface antigen, also referred to as the late HBsAg polypeptide. PreS.sub.1 consists of between 389-400 amino acids (depending on the antigenic subtype of HBV). The sequence unique to PreS.sub.1 consists of 108-119 amino acids which is added to the N-terminus of the complete PreS.sub.2 protein. The PreS.sub.1 protein is approximately 43 kilodaltons and may also be referred to as the P43 protein. PreS.sub.1 also exists in a glycosylated form of 46 kilodaltons designated as GP46 glycoprotein.
In the course of an HBV infection complete vital nucleocapsids are enveloped in a lipoprotein coat, forming 42 nm particles. Also formed during the HBV infection are empty 22 nm particles which consist mostly of the S and preS.sub.2 proteins, and some preS.sub.1 proteins. While the complete viral nucleocapsid is infectious, the 22 nm empty particles are not infectious. The empty particles, however, will elicit an immune response sufficient to confer immunity and may be used in the preparation of vaccines to HBV.
Hepatitis B vaccines prepared with 22 nm particles historically were prepared from the plasma of human carriers of KBV. Unfortunately 22 nm particles derived from human plasma must be extensively purified to remove infectious HBV particles as well as any other plasma-borne pathogens. Additionally the preparation of hepatitis B vaccine has been severely restricted because of the limited availability of human plasma.
Utilizing recombinant DNA biotechnology, it has been possible to express the hepatitis S protein in a 22 nm particle in transformed for example mammalian cell lines, and Saccharomyces cerevisiae. The mammalian systems currently utilized are expensive to use and the Saccharomyces systems produce relatively low yields of S protein.
Efforts to produce the antigenic and potentially more vaccine-effective PreS.sub.2 protein have proven unusually difficult. The PreS.sub.2 protein has been found to be very susceptible to proteolysis in recombinant systems. Proteolysis yields two smaller protein fragments which may not retain PreS.sub.2 's antigenicity. Additionally, the PreS.sub.2 protein has been very difficult to express in recombinant systems. The expression level of PreS.sub.2 is approximately 1/10 th the level of the S protein produced in the same recombinant systems.
It would be a significant contribution to the art to develop an enhanced process for the production of antigenic HBV particles containing the S protein and PreS.sub.2 protein of HBV. These particles would combine the major S protein with the more potentially antigenic PreS.sub.2 protein in a potentially more vaccine-effective form.
Therefore, it is an object of this invention to provide a process for the enhanced production of antigenie HBV particles consisting essentially of S protein and PreS.sub.2 protein of HBV.
Yet another object of this invention is to provide novel vectors containing DNA sequences which code for S protein and PreS.sub.2 protein.
A further object of this invention is to provide novel methylotrophic yeasts transformed with a vector or vectors capable of enhanced production of the HBV particle consisting of the S protein and an unglycosylated PreS.sub.2 protein.
Still another object of this invention is the product produced by the process for the production of antigenic HBV particle consisting essentially of S protein and PreS.sub.2 protein.
These and other objects of the invention will become apparent from the disclosure and claims herein provided.